Hon S. Ip,1 Mia Kim Torchetti,1 Rocio Crespo, Paul Kohrs, Paul DeBruyn, Kristin G. Mansfield,

Timothy Baszler, Lyndon Badcoe, Barbara Bodenstein, Valerie Shearn-Bochsler,

Mary Lea Killian, Janice C. Pedersen, Nichole Hines, Thomas Gidlewski,

Thomas DeLiberto, Jonathan M. SleemanNovelEurasianlineageavianinfluenzaA(H5N8)virushasspreadrapidlyandgloballysinceJanuary2014.InDecem-ber2014,H5N8andreassortantH5N2virusesweredetect-ed inwildbirds inWashington,USA,andsubsequently inbackyardbirds.Whentheyinfectcommercialpoultry,thesehighly pathogenic viruses pose substantial trade issues.

The novel Eurasian lineage clade 2.3.4.4 highly patho-genic avian influenza (HPAI) A(H5N8) virus (http://

www.who.int/influenza/gisrs_laboratory/h5_nomencla-ture_clade2344/en/) spread rapidly and globally during 2014, substantially affecting poultry populations. The first outbreaks were reported during January 2014 in chickens and domestic ducks in South Korea and subsequently in China and Japan (1–4), reaching Germany, the Nether-lands, and the United Kingdom by November 2014 and Italy in early December 2014 (5). Also in November 2014, a novel HPAI H5N2 virus was reported in outbreaks on chicken and turkey farms in Fraser Valley, British Colum-bia, Canada (5). This H5N2 influenza virus is a reassortant that contains the Eurasian clade 2.3.4.4 H5 plus 4 other Eurasian genes (polymerase acidic protein subunit, matrix protein, polymerase basic protein subunit [PB] 2, nonstruc-tural protein) and 3 North American wild bird lineage genes (neuraminidase [NA], nucleoprotein, PB1) (5). Taiwan has recently reported novel reassortants of the H5 clade 2.3.4.4 with other Eurasian viruses (H5N2, H5N3).

The appearance of highly similar Eurasian H5N8 vi-ruses in Asia, Europe, and now the United States suggests that this novel reassortant may be well adapted to certain waterfowl species, enabling it to survive long migrations (6). These appearances also represent a major change in Eurasian H5 virus circulation. After the reported spread of HPAI H5N1 virus in Asia, a large, interagency avian in-fluenza virus (AIV) surveillance effort was implemented throughout the United States during April 2006–March 2011 (7). Of nearly 500,000 wild bird samples tested, none harbored Eurasian subtype H5 AIV. The overall prevalence of AIV was ≈11%, and most viruses (86%) were detected in dabbling ducks (family Anatidae) (8). Although H5N8 subtype viruses have been detected previously in the Unit-ed States, all have been low pathogenicity AIV of North American wild bird lineage.

Case ReportsAfter the November 2014 report of H5N2 HPAI outbreaks among poultry in British Columbia, the US Departments of Agriculture and Interior, together with state agency personnel, increased surveillance of poultry flocks, hunt-er-harvested wild birds, and wild bird die-offs along the US–Canada border. A wild bird die-off was reported on December 1, 2014, at Wiser Lake (48.9039N, 122.4799W) in Whatcom County, Washington, USA. The lake, which has a history of waterfowl deaths caused by lead poisoning and aspergillosis, is located ≈32 km from the location of the index cases in Fraser Valley. Up to 10,000 waterfowl were on the lake when the deaths began. The dead birds con-sisted primarily of mallards (Anas platyrhynchos), Ameri-can wigeon (A. americana), and northern pintail (A. acuta), along with smaller numbers of other waterfowl species.

Nine carcasses were submitted to the National Wild-life Health Center (Table 1); 6 were examined in detail. Aspergillus fumigatus was isolated from 5 birds with char-acteristic lesions of airsacculitis (Table 1). In addition, cloacal and/or oral swab samples from 5 birds had molec-ular assay results positive for influenza A and H5 (Table 1). A Eurasian lineage H5 clade 2.3.4.4 AIV, A/northern pintail/Washington/40964/2014 (H5N2) (GenBank taxon no. 1589662), was isolated from a lung specimen. Whole-genome sequencing indicated the virus was highly similar to the H5N2 reassortant virus from Canada. Both viruses have 3 RNA segments of North American wild bird lineage

Novel Eurasian Highly Pathogenic Avian Influenza A H5 Viruses in Wild Birds,

Washington, USA, 2014

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Authoraffiliations:USGeologicSurvey–NationalWildlifeHealthCenter,Madison,Wisconsin,USA(H.S.Ip,B.Bodenstein, V.Shearn-Bochsler,J.M.Sleeman);USDepartmentofAgriculture,Ames,Iowa,USA(M.K.Torchetti,M.L.Killian,J.C.Pedersen, N.Hines);WashingtonStateUniversity,Pullman,Washington,USA(R.Crespo,T.Baszler);WashingtonDepartmentof Agriculture,Olympia,Washington,USA(P.Kohrs,T.Baszler, L.Badcoe);WashingtonDepartmentofFishandWildlife,Olympia(P.DeBruyn,K.G.Mansfield);USDepartmentofAgriculture, FortCollins,Colorado,USA(T.Gidlewski,T.DeLiberto)

DOI:http://dx.doi.org/10.3201/eid2105.1420201Thesefirstauthorscontributedequallytothisarticle.

HPAIAH5VirusesinBirds,Washington,USA

(PB1, nucleoprotein, and NA) and 5 RNA segments (PB2, polymerase acidic, HA, matrix protein, and nonstructural protein) that showed >99% similarity to 2014 Eurasian clade 2.3.4.4 H5N8 viruses (Table 2). According to World Organisation for Animal Health guidelines (9), the virus was consistent with HPAI on the basis of the amino acid sequence at the hemagglutinin cleavage site and in vivo as-say results (intravenous pathogenicity index 2.57).

In a related event, on December 6, 2014, an American wigeon was captured and partially consumed by a captive-reared gyrfalcon (Falco rusticolus) in Whatcom County, ≈8 km from Wiser Lake. The wigeon remains were also fed to 3 other gyrfalcon and gyrfalcon–peregrine hybrids at a farm with another 25 raptors and 40 pigeons. The first falcon (RW099878) died on December 8 and was submit-ted to the National Wildlife Health Center. The second fal-con (RX085847) also died on December 8 and the third (RX093091) on December 11; the fourth (RX084955) was euthanized on December 11. Carcasses of these last 3 fal-cons were submitted to the Washington Animal Disease Diagnostic Laboratory. No further deaths or illnesses have been reported among other raptors at the facility. Histologic

and pathologic findings for the 3 raptors were consistent with those described in previous reports of H5N8 infections (1,6), and the severity of the lesions corresponded to virus concentrations detected in the tissues by molecular assays; results will be further detailed in a subsequent publication.

Molecular assay results for oral and cloacal swab samples and major organ and brain samples from falcon RW099878 were positive for influenza A and H5 viruses. For falcons RW099878 and RX085847, partial HA and NA genes were directly sequenced from brain and oral swab samples. A Eurasian lineage H5 clade 2.3.4.4 AIV, A/gyrfalcon/Washington/41088-6/2014 (H5N8) (GenBank taxon no. 1589663), was isolated from the brain of falcon RW099878. All 8 RNA segments for the strain were >99% similar to those for 2014 Group A H5N8 strains from South Korea (3) (Table 2); the amino acid sequence at the hemag-glutinin cleavage site and in vivo assay results (intravenous pathogenicity index 2.65) were consistent with HPAI.

Phylogenetic analysis of the H5 clade 2.3.4.4 viruses detected in the United States resulted in 3 major findings (Figures 1, 2; online Technical Appendix, http://wwwnc.cdc.gov/EID/article/21/5/14-2020-Techapp1.pdf). First, the

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Table 1. Summary of influenza test results for samples from 10 birds from Washington,USA,2014* Sample ID no. Species,commonname Necropsy AIV status Diagnostic finding 26080–001 Northernshoveler Yes PCRnegative,isolationnegative Aspergillosis 26080–002 Northernpintail Yes SubtypeH5N2 Aspergillosis,HPAI 26080–003 American wigeon Yes PCRpositive,isolationnegative Aspergillosis 26080–004 American wigeon No PCRnegative,isolationnegative ND 26080–005 American wigeon No PCRnegative,isolationnegative ND 26080–006† Mallard Yes PCRpositive,isolationnegative Aspergillosis 26080–007† Mallard Yes PCRpositive,isolationnegative Aspergillosis 26080–008† Mallard No PCRpositive,isolationnegative ND 26080–009 Trumpeter swan Yes PCRnegative,isolationnegative Emaciation RW099878 Gyrfalcon Yes SubtypeH5N8 HPAI *AIV,avianinfluenzavirus;HPAI,highlypathogenicavianinfluenza;ID,identification,ND,notdetermined. †Specimens tested by the Washington Animal Disease Diagnostic Laboratory.

Table 2. NucleotideidentitybetweentheinfluenzaA(H5N2)andA(H5N8)virusesfromWashington,USA,andtheirnearesthomologuesinGenBankasofJanuary8,2015* Virus from Washington State Nearesthomologue % Identity A/Northernpintail/Washington/40964/2014(H5N2) PB2 A/beangoose/Korea/H40/2014 99.5 PB1 A/bufflehead/California/3118/2011 99.0 PA A/commonteal/Korea/H455–30/2014 99.5 HA A/crane/Kagoshima/KU1/2014 99.3 NP A/American green-winged teal/Ohio/13OS2084/2013 99.1 NA A/bufflehead/California/4935/2012 99.0 MP A/Baikalteal/Korea/S005/2014 100.0 NS A/Baikalteal/Korea/Donglim3/2014 99.9

A/gyrfalcon/Washington/40188–6/2014(H5N8) PB2 A/bean goose/Korea/H40/2014 99.6 PB1 A/Baikalteal/Korea/H41/2014 99.4 PA A/Baikalteal/Korea/Donglim3/2014 99.3 HA A/crane/Kagoshima/KU1/2014 99.2 NP A/Baikalteal/Korea/H41/2014 99.5 NA A/Coot/Korea/H81/2014 99.4 MP A/Baikalteal/Korea/Donglim3/2014 99.9 NS A/Baikalteal/Korea/Donglim3/2014 100.0

*HA,hemagglutinin;MP,matrixprotein;NA,neuraminidase;NP,nucleoprotein;NS,nonstructural,PA,polymeraseacidic;PB1and2,polymerasebasic1and 2.

DISPATCHES

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Figure 1.PhylogeneticcomparisonofthecompletehemagglutiningenesofhighlypathogenicavianinfluenzaA(H5N2)andA(H5N8)strainsfromtheUnitedStateswithstrainsfromAsia,Europe,andCanada.SolidcirclesindicateH5N2andH5N8strainsfromtheUnitedStates;opencircleindicatesH5N2strainfromCanada;blacktriangleindicatesH5N8strainfromacraneinJapan.SequenceswerealignedbyusingMUSCLE,andphylogeneticandmolecularevolutionaryanalyseswereconductedbyusingMEGAversion5,usingtheneighbor-joiningtree-buildingmethod,with1,000bootstrapreplicates(10). Analysis was done with viruses that were phylogenetically representativeofappropriatelineages(InfluenzaVirusResourceDatabase,http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).Scalebar indicates nucleotide substitutions per site.

HPAIAH5VirusesinBirds,Washington,USA

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Figure 2. PhylogeneticcomparisonofthecompleteneuraminidasegenesofhighlypathogenicavianinfluenzaA(H5N2)(panelA)andA(H5N8)(panelB)strainsfromtheUnitedStateswithstrainsfromAsia,Europe,andCanada.SolidcirclesindicateH5N2andH5N8strainsfromtheUnitedStates;blacktriangleindicatesH5N8strainfromacraneinJapan.SequenceswerealignedbyusingMUSCLE,andphylogeneticandmolecularevolutionaryanalyseswereconductedbyusingMEGAversion5,usingtheneighbor-joiningtree-buildingmethod,with1,000bootstrapreplicates(10). Analysis was done with viruses that were phylogenetically representative of appropriatelineages(InfluenzaVirusResourceDatabase,http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).Scalebarindicatesnucleotide substitutions per site.

DISPATCHES

Eurasian lineage avian H5N8 clade 2.3.4.4 virus survived introduction into North America in its entirety. Second, in-troduction of Eurasian H5N8 virus into North America ap-pears to be independent from introductions of the virus into Europe. Third, the duration of circulation of H5N8 virus in the Pacific flyway (California, Idaho, Nevada, Oregon, Utah, and Washington, USA) is unknown, but it was suffi-cient for reassortment with low pathogenicity North Ameri-can lineage wild bird AIV (Figure 1).

ConclusionsThe ongoing circulation of these Eurasian HPAI H5 virus-es in wild birds considerably alters the potential risks and subsequent consequences for US poultry and wildlife re-habilitation centers. Detection of HPAI H5N8 virus in ap-parently healthy common teal (A. crecca), Eurasian wigeon (A. penelope), mallard, spot-billed duck (A. poecilorhyn-cha), and tundra swans (C. columbianus) (3,5) suggests that wild birds may contribute to further spread of this HPAI H5 lineage in North America. However, culling and oth-erwise disturbing wild birds or their habitats has not been shown to be beneficial in the control of avian influenza (11). The scientifically supported management action (11) is to enhance biosecurity to minimize contacts between poul-try, wild birds, and their fomites (11). In addition, hunters should be cognizant of risks from handling potentially in-fected carcasses (http://www.aphis.usda.gov/animal_health/ birdbiosecurity/downloads/USDA_HntrCd_Hi.pdf).

Examination of wild bird surveillance samples col-lected before December 2014 may provide further insight into the timing and route of introduction of the Eurasian clade 2.3.4.4 H5N8 virus into North America. In addition, enhanced and ongoing influenza surveillance in wild birds and poultry will contribute to a better understanding of the geographic distribution and species involved in the spread of these HPAI H5 viruses. Together, these data may enable waterfowl managers and poultry producers to better as-sess and manage disease risks. Human infections have not been associated with either virus; however, H5 clade 2.3.4 H5N1 virus has caused human death, so caution is warrant-ed. During preparation of this article, H5N8 was reported in wild birds and poultry along the Pacific flyway; novel H5N2 virus was detected in Idaho, Oregon, and Washing-ton; and another novel reassortant H5N1 was detected in Washington and British Columbia (5). These detections have had major effects on US poultry trade (12).

AcknowledgmentsWe acknowledge the dedicated staff at our respective institu-tions, who ensured timely field support, rapid response, and prompt and accurate laboratory testing; in particular, Renee Long, Katy Griffin, Jeffery Messer, Karin McMullen, Amber Itle, and Thomas Gilliom.

Dr. Ip is the section head of the Diagnostic Virology Laboratory, Wildlife Disease Diagnostic Laboratories Branch, US Geologic Survey–National Wildlife Health Center. His research interests include emerging diseases of wildlife, disease transmission and persistence in natural populations, and laboratory diagnostics de-velopment.

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Address for correspondence: Mia Kim Torchetti, USDA APHIS VS National Services Veterinary Laboratories, 1920 Dayton Ave, Ames, IA 50010, USA; email: Mia.Kim.Torchetti@aphis.usda.gov

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